1. Field of the Invention
The invention is directed to methods for identifying compounds that inhibit or prevent infection of cells by enveloped viruses such as HIV-1, and the compounds discovered by such methods. The invention also includes using these methods as diagnostic assays to detect antibodies in virus-infected individuals that inhibit the viral entry processes.
2. Related Art
The HIV-1 envelope glycoprotein is a 160 kDa glycoprotein that is cleaved to form the transmembrane (TM) subunit, gp41, which is non-covalently attached to the surface (SU) subunit, gp120 (Allan J. S., et al., Science 228:1091-1094 (1985); Veronese F. D., et al., Science 229:1402-1405 (1985)). Recent efforts have led to a clearer understanding of the structural components of the HIV-1 envelope system. Such efforts include crystallographic analysis of significant portions of both gp120 and gp41 (Kwong, P. D., et al., Nature (London) 393:648-659 (1998); Chan, D. C., et al., Cell 89:263-273 (1997); Weissenhorn, W., et al., Nature 387:426-430 (1997)).
The surface subunit has been characterized as part of a multi-component complex consisting of the SU protein (the gp120 core absent the variable loops) bound to a soluble form of the cellular receptor CD4 (N-terminal domains 1 and 2 containing amino acid residues 1-181) and an antigen binding fragment of a neutralizing antibody (amino acid residues 1-213 of the light chain and 1-229 of the heavy chain of the 17b monoclonal antibody) which blocks chemokine receptor binding (Kwong, P. D., et al., Nature (London) 393:648-659 (1998)). Several envelope components believed to exist only in the fusion-active form of gp120 were revealed by the crystallographic analysis including a conserved binding site for the chemokine receptor, a CD4-induced epitope and a cavity-laden CD4-gp120 interface. This supports earlier observations of CD4-induced changes in gp120 conformation.
The gp120/gp41 complex is present as a trimer on the virion surface where it mediates virus attachment and fusion. HIV-1 replication is initiated by the high affinity binding of gp 120 to the cellular receptor CD4 and the expression of this receptor is a primary determinant of HIV-1 cellular tropism in vivo (Dalgleish, A. G., et al., Nature 312:763-767 (1984); Lifson, J. D., et al., Nature 323:725-728 (1986); Lifson, J. D., et al., Science 232:1123-1127 (1986); McDougal, J. S., et al., Science 231:382-385 (1986)). The gp120-binding site on CD4 has been localized to the CDR2 region of the N-terminal V1 domain of this four-domain protein (Arthos, J., et al., Cell 5:469-481 (1989)). The CD4-binding site on gp120 maps to discontinuous regions of gp120 including the C2, C3 and C4 domains (Olshevsky, U., et al., Virol 64:5701-5707 (1990); Kwong, P. D., et al., Nature (London) 393:648-659 (1998)). Following attachment to CD4, the virus must interact with a xe2x80x9csecondxe2x80x9d receptor such as a chemokine receptor in order to initiate the fusion process. Recently, researchers have identified the critical role of members of the chemokine receptor family in HIV entry (McDougal J. S., et al., Science 231:382-385 (1986); Feng Y., et al., Science 272:872-877 (1996); Alkhatib G., et al., Science 272:1955-1958 (1996); Doranz B. J., et al., Cell 85:1149-1158 (1996); Deng H., et al., Nature 381:661-666 (1996); Dragic T., et al., Nature 381:667-673 (1996); Choe H., et al., Cell 85:1135-1148 (1996); Dimitrov D. S., Nat. Med. 2:640-641 (1996); Broder, C. C. and Dimitrov, D. S., Pathobiology 64:171-179 (1996)). CCR5 is the chemokine receptor used by macrophage-tropic and many T-cell tropic primary HIV-1 isolates. Most T-cell line-adapted strains use CXCR4, while many T-cell tropic isolates are dual tropic, capable of using both CCR5 and CXCR4.
Binding of gp120 to CD4 and a chemokine receptor initiates a series of conformational changes within the HIV envelope system (Eiden, L. E. and Lifson, J. D., Immunol. Today 13:201-206 (1992); Sattentau, Q. J. and Moore J. P., J. Exp. Med. 174:407-415 (1991); Allan J. S., et al., AIDS Res Hum Retroviruses 8:2011-2020 (1992); Clapham, P. R., et al., J. Virol. 66:3531-3537 (1992)). These changes occur in both the surface and transmembrane subunits and result in the formation of envelope structures which are necessary for virus entry. The functions of gp41 and gp120 appear to involve positioning the virus and cell membranes in close proximity thereby facilitating membrane fusion (Bosch M. L., et al., Science 244:694-697 (1989); Slepushkin, V. A. et al., AIDS Res Hum Retroviruses 8:9-(1992); Freed E. O. et al., Proc. Natl. Acad. Sci. USA 87:4650-4654 (1990)).
A good deal of structural information is available with respect to the HIV-1 transmembrane glycoprotein (gp41). This protein contains a number of well-characterized functional regions. See FIG. 3. For example, the N-terminal region consists of a glycine-rich sequence referred to as the fusion peptide which is believed to function by insertion into and disruption of the target cell membrane (Bosch, M. L., et al., Science 244:694-697 (1989); Slepushkin, V. A., et al., AIDS Res. Hum. Retrovirus 8:9-18 (1992); Freed, E. O., et al., Proc. Natl. Acad. Sci. USA 87:4650-4654 (1990); Moore, J. P., et al., xe2x80x9cThe HIV-cell Fusion Reaction,xe2x80x9d in Viral Fusion Mechanism, Bentz, J., ed., CRC Press, Inc., Boca Raton, Fla.). Another region, characterized by the presence of disulfide linked cysteine residues, has been shown to be immunodominant and is suggested as a contact site for the surface (gp120) and transmembrane glycoproteins (Gnann, J. W., Jr., et al., J. Virol. 61:2639-2641 (1987); Norrby, E., et al., Nature 329:248-250 (1987); Xu, J. Y., et al., J. Virol. 65:4832-4838 (1991)). Other regions in the gp41 ectodomain have been associated with escape from neutralization (Klasse, P. J., et al., Virology 196:332-337 (1993); Thali, M., et al., J. Virol. 68:674-680 (1994); Stem, T. L., et al., J. Virol. 69:1860-1867 (1995)), immunosuppression (Cianciolo, G. J., et al., Immunol. Lett. 19:7-13 (1988); Ruegg, C. L., et al., J. Virol. 63:3257-3260 (1989)), and target cell binding (Qureshi, N. M., et al., AIDS 4:553-558 (1990); Ebenbichler, C. F., et al., AIDS 7:489-495 (1993); Henderson, L. A. and Qureshi, M. N., J. Biol. Chem. 268:15291-15297 (1993)).
Recent work has increased knowledge of the structural components of the HIV-1 transmembrane glycoprotein, however, the immunogenic nature of gp41 remains poorly understood. It is known that one of two immunodominant regions present in the HIV-1 envelope complex is located in gp41 (Xu, J. Y., et al., J. Virol. 65:4832-4838 (1991)). This region (TM residues 597-613) is associated with a strong, albeit non-neutralizing, humoral response in a large number of HIV+ individuals.
Two regions of the ectodomain of gp41 have been shown to be critical to virus entry. Primary sequence analysis predicted that these regions (termed the N-helix (residues 558-595 of the HIV-1LAI sequence) and C-helix (residues 643-678 of the HIV-1LAI sequence)) model the xcex1-helical secondary structure. Experimental efforts stemming from previous structural studies of synthetic peptide mimics established that the sequence analysis predictions were generally correct (Wild, C., et al., Proc. Natl. Acad. Sci. USA 89:10537-10541 (1992); Wild, C. T., et al., Proc. Natl. Acad. Sci. USA 91:9770-9774 (1994); Gallaher, W. R., et al., AIDS Res. Hum. Retroviruses 5:431-440 (1989); Delwart, E. L., et al., AIDS Res. Hum. Retroviruses 6:703-704 (1990)). Subsequent structural analysis determined that these regions of the transmembrane protein interact in a specific fashion to form a higher order structure characterized as a trimeric six-helix bundle (Chan, D. C., et al., Cell 89:263-273 (1997); Weissenhorn, W., et al., Nature 387:426-430 (1997)). This trimeric structure consists of an interior parallel coiled-coil trimeric core (region one, N-helix) which associates with three identical xcex1-helices (region two, C-helix) which pack in an oblique, antiparallel manner into the hydrophobic grooves on the surface of the coiled-coil trimer. This hydrophobic self-assembly domain is believed to constitute the core structure of gp41. See FIGS. 4A and 4B. It has been demonstrated that the N-and C-helical regions of the transmembrane protein are critical to HIV-1 entry. It has been proposed that the association of these two regions to form the six-helix bundle core structure occurs during the transition from a nonfusogenic to a fusion-active form of gp41, and that the formation of this core structure facilitates membrane fusion by bringing the viral and target cell surfaces into close proximity (Chan, D. C. and Kim, P. S., Cell 93:681-684 (1998); FIG. 1). If correct, the formation of the six-helix bundle is a key step in virus entry and factors which interfere with its formation could disrupt the entry event. A number of viruses share protein glycoprotein structure similar to N- and C-helical regions of HIV transmembrane protein (Lambert et al., Proc. Nat. Acad Sci. 93:2186-2191 (1996). See also, Published PCT Application No. WO96/19495.
All approved drugs for the treatment of human immunodeficiency virus (HIV) infection target either viral reverse transcriptase (RT) or protease activity. Although certain combinations of these drugs have proven highly effective in suppressing virus replication, problems related to complicated dosing regimens and selection for resistant viral isolates necessitate the continued need for the development of additional therapies. To maximize their effect in combination therapy these new drugs should exploit targets other than RT or protease.
Mono- and bi-therapy for human immunodeficiency virus type 1 (HIV-1) infection are only transiently effective mainly due to virus drug resistance. To obtain a sustained benefit from antiviral therapy, current guidelines recommend at least triple-drug combinations, or the so-called highly active antiretroviral therapy (HAART). Despite these advances, there are still problems with the currently available drug regimens. Many of the drugs exhibit severe toxicities or require complicated dosing schedules that reduce compliance and limit efficacy. Resistant strains of HIV usually appear over extended periods of time even on HAART regimens.
For these and other reasons there is a continuing need for the development of additional anti-HIV drugs. Ideally these would target different stages in the viral life cycle, (adding to the armamentarium for combination therapy), exhibit minimal toxicity, and have low manufacturing costs. Small molecule inhibitors of HIV entry could aid significantly in addressing these problems.
It has been proposed that the DP-107 and DP-178 peptides inhibit HIV-1 replication by disrupting formation of the six-helix bundle in a negative-dominant manner (FIG. 2). As prototypes of a new class of HIV inhibitors which block virus entry, these compounds offer additional therapeutic options for use alone or in combination with drugs targeting other steps in virus replication. However, as is often the case with protein-based therapeutics, these peptides are less than ideal drug candidates due to issues of oral bioavailability, in vivo stability and manufacturing costs.
The 2F5 monoclonal antibody, from isolates presenting the gp41 sequence ELDKWAS, is a neutralizing antibody targeting gp41 (Muster, T., et al. J. Virol. 67:6642-6647 (1993), and Muster, T., et al., J. Virol. 68:4031-4034 (1994)). This antibody maps to the linear amino acid sequence Glu-Leu-Asp-Lys-Trp-Ala (ELDKWA) in the ectodomain of gp41, an epitope which is conserved in 72% of HIV-1 isolates. While this antibody maps to a linear determinant, competition studies suggest that the 2F5 epitope is conformational in nature.
The monoclonal antibody, NC-1 has been shown to bind the six-helix bundle in fusion-active gp41 (Jiang, S., et al., J. Virol. 72:10213-10217 (1998)). NC-1 was generated and cloned from a mouse immunized with a mixture of peptides modeling the N- and C-helical domains of gp41. NC-1 binds specifically to both the xcex1-helical (N-helical) core domain and an oligomeric form of gp41. This conformation-dependent reactivity is dramatically reduced by point mutations within the N-terminal coiled-coil region of gp41 which impede formation of the six helix bundle. NC-1 binds to the surfaces of HIV-1-infected cells only in the presence of soluble CD4.
Formaldehyde-fixed, fusion active whole-cell preparations (in transgenic mice) have been used to generate an antisera capable of neutralizing 23 of 24 primary HIV isolates from diverse geographic locations and genetic clades A to E (LaCasse, R. A., et al., Science 283:357-362 (1999)). These fusion-competent immunogens may capture the transient envelope-CD4-co-receptor structures that arise during HIV binding and fusion.
A number of viruses share similar protein/glycoprotein structures which have been implicated in the mechanism of viral fusion and entry into permissive cells. The present invention provides methods of screening for compounds that inhibit viral fusion and/or entry into permissive cells. The screening methods of the invention involve attempting to selectively trigger the formation of one or more critical entry intermediates in cell-surface-expressed viral envelope in the presence of a test compound and probing for the formation or lack of formation of such intermediates. This can be accomplished as described herein.
A specific embodiment of the invention is directed to a method for determining compounds which disrupt formation of critical gp41 structures and conformations necessary for virus entry and therefore block HIV entry. The gp41 six-helix bundle which forms in response to CD4/gp120 binding constitutes one such critical entry structure. Antibodies specific for the six-helix bundle are used to determine the ability of small molecules to block its formation. The method of the present invention can be applied to other viruses where a transmembrane protein or glycoprotein forms structures and complexes that are involved for virus entry, including but not limited to, HIV-2, HTLV-I, HTLV-II, respiratory syncytial virus (RSV), human influenza viruses, parainfluenza virus type 3 (HPIV-3), Newcastle disease virus, feline immuno-deficiency virus (FIV), and measles virus.
The invention is also directed to novel inhibitors identified by these methods, which can be small molecules, peptides, proteins, antibodies and antibody fragments, or derivatives thereof. These inhibitors are suitable for inhibiting or preventing infection by various viruses including HIV-1 and/or the other viruses listed above. These inhibitors can be used to treat humans infected with HIV-1 or the other viruses, or used to prevent infection by HIV-1 or the other viruses. The invention also includes the inhibitors in suitable pharmaceutical compositions.
Compounds that show inhibitory activity in the assays of the current invention may act at any of the several steps leading to, or associated with, the conformational changes in the viral envelope glycoproteins that result in membrane fusion. For example they may inhibit the interaction between the envelope glycoprotein and its receptors which are the triggers that initiate conformation changes in the envelope glycoproteins (e.g. in the case of HIV-1, the interaction between gp120 and CD4 or the CCR5 or CXCR4 chemokine receptors). Alternatively, they may directly inhibit the formation of fusion active structures, e.g. by preventing the association of the alpha helical domains of the transmembrane protein that are part of these structures (e.g. in the case of HIV-1, by blocking the association of the N- and C-helical domains that lead to six helix bundle formation). The assays are also capable of discovering inhibitors of other steps in the process that are as yet not fully elucidated.
Additional assays can be performed to analyze in more detail the mechanism of action of inhibitory compounds discovered in the present invention. The methods for these assays are well know to those skilled in the art. For example, assays to test inhibitors of the HIV-1 gp120 interaction with CD4 or chemokine receptors are described in Dragic, T., et al., Nature 381:667-673 (1996) and Donzella, G. A., et al., Nature Medicine 4:72-77 (1998). Assays to test inhibitors of HIV-1 gp41 6 helix bundle formation are described in Jiang S. et al., J. Virol. Methods 80:85-96 (1999).
This invention also includes the use of the assays described above as diagnostic assays to detect antibodies in virus-infected individuals or virus-infected body fluids or tissues that inhibit entry-relevant conformational changes in one or more viral envelope proteins or glycoproteins. The presence of such antibodies in infected individuals or samples is of prognostic value.